937. siRNA Inhibition of MMP9 Expression Inhibits TGFβ1-Induced Calcification of Aortic Valve Interstitial Cells

Abstract

The leading cause of both native and bioprosthetic heart valve failure is calcification, which has been linked to the expression of bone-related proteins and matrix metalloproteinases (MMP), and in particular MMP9. Previous findings in our laboratory have shown that TGFβ1 mediates calcification through an apoptotic mechanism. Furthermore, we have discovered TGFβ1 induces MMP9 expression in a time-dependent fashion in cell culture. In addition, strong positive MMP9 immunohistochemical staining in calcified aortic valves compared to minimal staining in noncalcified, bicuspid and tricuspid regurgitant valves further demonstrated MMP9 involvement in the calcification process. However, its defined role in the pathway still remains unclear. Our hypothesis is that MMP9 regulates TGFβ1 activation, ultimately influencing the calcification process in aortic valve interstitial cells. We designed small interfering RNA, siRNAs, for MMP9, targeted at both the catalytic and fibronectin sites. Zymography showed that siRNA was effective in downregulation of TGFβ1-induced MMP9 expression. The reduction of MMP9 resulted in decreased apoptosis, associated with initiation of calcification, based on negligible annexin V staining in DAPI-localized cells compared to control. Therefore, these results indicate that MMP9 may be involved in the calcification progression by influencing the activation and mechanism(s) exerted by TGFβ1.